Efficient and accurate protein secretion is a fundamental process that plays a pivotal role in the life of all eukaryotic cells. Fully one-third of the eukaryotic proteome is targeted to the membrane compartments that comprise the secretory pathway. These proteins must be faithfully delivered to these organelles after synthesis and folding in the endoplasmic reticulum (ER). The first step in protein delivery to downstream compartments is the selective capture of proteins into ER-derived transport vesicles, known as COPII vesicles for the cytoplasmic coat proteins that sculpt these transport carriers from the donor membrane. Cargo proteins are enriched in nascent vesicles through the action of the COPII coat subunit, Sec24p, which serves as a cargo- binding platform by providing multiple interfaces for cargo-coat interaction. Although we have gained enormous insight into this function of Sec24p through biochemical, genetic and structural analyses, we are currently lacking a full description of the diversity and flexibility of this process, which must accommodate a vast array of client proteins with distinct structures, functions, and ultimate destinations. We aim to fully understand the molecular interactions that drive export out of the ER, and are working to determine the full spectrum of cargo proteins that bind to Sec24p to ensure efficient ER egress. We use the model organism, Saccharomyces cerevisiae, to study this fundamental process using a combination of genetic, proteomic and biochemical approaches. This research proposal consists of two specific aims. (1) To determine the full repertoire of secretory proteins that interacts with the three known cargo-binding sites on Sec24p and to define the mode of interaction of novel client cargoes. (2) To determine the nature of cargo capture or coat assembly defects associated with three novel Sec24p mutants that were isolated from a structure-based genetic screen. Using these approaches, we seek to gain detailed insight into the molecular mechanisms that underlie the diversity and flexibility of cargo capture. Ultimately, a more detailed understanding of this fundamental eukaryotic process will have important implications in the many aspects of human disease and development that are impacted by early stages of protein biogenesis and deployment within the secretory pathway. We study the detailed molecular interactions that drive the selective capture of newly synthesized secretory proteins into transport vesicles that bud from the endoplasmic reticulum. Public Health Relevance: This process represents a critical quality control decision in the cell, and has important implications for the growing number of diseases that are caused by aberrant folding, trafficking and deployment of secretory proteins, including cystic fibrosis, familial heart disease and lung disease. [unreadable] [unreadable] [unreadable] [unreadable] [unreadable] [unreadable]